The long-range goal is to define morphological, structural, and functional interactions among cells of the fetal membranes that contribute to the maintenance of the physical integrity of this tissue. Selected studies of this project are coordinated with those designed to define the ontogeny of interstitial pro-collagen synthesis/processing in amnion. An alternative approach to most previous studies is proposed; viz, an investigation of the region of the fetal membranes most prone to rupture, i.e., the membranes immediately over the internal cervical os ("dependent" membranes) prior to membrane rupture. Focusing on the amnion, the cellular and structural changes that may be involved in spontaneous rupture during labor and those that may create greater vulnerability for premature rupture (PT-PROM) will be defined. The amnion is an important physical/physiological barrier. but in some pregnancies during labor, leukocytes migrate into and sometimes through the fetal membranes to accumulate in the amniotic fluid (AF). Yet, surprisingly little is known about this process, including the origin of the cells, and the route and mechanisms of transmigration. We propose 3 specific aims. The goal of Aim 1 is to define, by anatomical and morphological analyses, the changes in cells, cellular relationships, and collagen structure in the amnion of the dependent membranes during gestation by immunohistochemistry, electron microscopy, and confocal laser scanning microscopy applied to both conventional sections and whole tissue examined en face. The goal of Aim 2 is to conduct similar analyses of changes related to labor, and in pregnancies in which there is increased risk of premature rupture, viz., maternal smoking. The goal of Aim 3 is to define the origin of leukocytes with the fetal membranes and AF, the capacity of these cells to produce inflammatory cytokines, and the role of the amnion in providing the chemotactic signals that promote leukocyte migration. Leukocytes in AF and tissues from pregnancies with male fetuses will be analyzed for the Y chromosome by in situ hybridization. Chemotaxis assays will be conducted to define the capacity of amnion tissue and isolated cells to produce chemokines in response to relevant stimuli (IL-1, LPS, phagocytosis, cadmium, and hypoxia) and the ability of leukocytes to transmigrate amnion tissue under various conditions. The findings of these studies should provide important new information to clarify selected aspects of amnion function for which there are significant clinical implications.